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Molecular Characterization of Human Parvovirus B19 Genotypes

2 and 3

Zhaojun Chen 1,2 , Wuxiang Guan 2, Fang Cheng 2, Aaron Yun Chen 2, and Jianming Qiu 2,*1Department of Clinical Laboratory, The Affiliated Hospital of Hangzhou Normal University,Hangzhou, Zhejiang Province, China 3100152Department of Microbiology, Molecular Genetics and Immunology, University of Kansas MedicalCenter, Kansas City, Kansas

AbstractWe have characterized the transcription profiles of parvovirus B19 (B19V) genotype-2 A6 and

genotype-3 V9 variants. The A6 RNA profile differs from that of the prototype B19V in both B19Vnon-permissive and permissive cells, whereas the overall profile of the V9 RNA in these cells issimilar to that of the prototype. A unique feature we have identified is that the genotype-2 A6 variantused only one splice acceptor to remove the first intron. We also demonstrated that the inverted terminal repeats (ITRs) of the prototype B19V support replication of the V9 genome, which producesinfectious virus, but not that of the A6 genome, in B19V-permissive cells. Similar to the proapoptoticnature of the prototype B19V large non-structural protein (NS1), the A6 and V9 NS1 proteins alsoare potent inducers of apoptosis in B19V-permissive cells.

Keywords

Parvovirus; B19; genotype; transcription; replication; apoptosis

IntroductionHuman parvovirus B19 (B19V) is the only parvovirus that has been confirmed to be pathogenicin humans until the discovery of human bocavirus (Allander et al., 2005). B19V causes a varietyof diseases, including erythema infectiosum (fifth disease) in children, acute or chronicarthropathy in adults, aplastic crisis in patients with chronic hemolytic anemia, persistentanemia in immunodeficient and immunocompromised patients, and fetal hydrops in pregnantwomen (Young and Brown, 2004). Recently a number of B19V variants were reported to varyextensively from the prototype B19V with respect to genomic sequence, exhibiting greater than 12% divergence versus the less than 2% divergence characteristic of previouslycharacterized prototype B19V isolates (Servant et al., 2002; Hokynar et al., 2002; Nguyen etal., 1999). Of these, the V9 variant, which was isolated from a patient with aplastic crisis,

diverged by about 12% sequence from prototype B19V isolates (Nguyen et al., 1999). The A6variant, which was isolated from an anemic HIV-positive patient, exhibited 12% divergence

*Corresponding author: Jianming Qiu, Department of Microbiology, Molecular Genetics and Immunology, University of Kansas MedicalCenter, Mail Stop 3029, 3901 Rainbow Blvd., Kansas City, KS 66160, Phone: (913) 588-4329, Fax: (913) 588-7295, [email protected]'s Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting

proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

NIH Public AccessAuthor ManuscriptVirology . Author manuscript; available in PMC 2010 November 25.

Published in final edited form as:Virology . 2009 November 25; 394(2): 276285. doi:10.1016/j.virol.2009.08.044.NI H

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from the prototype B19V, and 8% divergence from the V9 variant (Nguyen et al., 2002). Thesequences of other reported A6-related isolates, including the LaLi isolate, were 98% identicalto that of the A6 variant (Hokynar et al., 2002). Therefore, the human erythrovirus is nowclassified into three distinct genotypes (Servant et al., 2002). Genotype-1 is composed of allthe prototype B19V isolates, genotype-2 includes the A6, LaLi and their related isolates(Blumel et al., 2005; Norja et al., 2006; Sanabani et al., 2006), and genotype-3 comprises theV9 and V9-related isolates (Sanabani et al., 2006; Candotti et al., 2004).

The B19V genome contains two identical terminal repeats (ITRs) of approximately 380nucleotides; these are imperfect palindromes and form hairpin loops. However, the terminalrepeats of genotype-2 and genotype-3 have not been cloned and sequenced. The genotype-1replicates restrictively in the erythroid progenitors of human bone marrow, often producingexceptionally high numbers of progeny virus in the blood (Ozawa et al., 1986; Srivastava and Lu, 1988). In contrast, high virus-load viremias of genotype-2 and genotype-3 have beenidentified only occasionally (Blumel et al., 2005; Nguyen et al., 1999; Nguyen et al., 2002;Servant et al., 2002; Liefeldt et al., 2005). However, both of these genotypes have beenassociated with anemia or aplastic crisis, indicating their tropism for erythroid cells (Blumelet al., 2005; Nguyen et al., 1999; Sanabani et al., 2006; Candotti et al., 2004). Cross-reactivityof 100% in antibody activities among these three genotypes supports the notion that theycomprise only a single serotype (Ekman et al., 2007). It is not yet known whether the variations

among the genomes of human erythrovirus variants are responsible for their distinct biologicaland pathological properties. The P6 promoter activities of the three genotypes have been shownto be identical, to be most pronounced in B19V-permissive cells (Ekman et al., 2007). However,the overall transcriptional profiles of the B19V genotype-2 and genotype-3 and their replicationcompetence with the genotype-1 ITRs have not been explored.

The disease outcome of B19V infection as seen in transient aplastic crisis, pure red cell aplasiaand hydrops fetalis is due to the direct cytotoxicity of the virus to the erythroid progenitors thatare native host of B19V replication (Brown and Young, 1997). It has been shown that duringB19V infection of primary erythroid progenitor cells and myeloid cell lines (Sol et al., 1999;Moffatt et al., 1998), e.g. UT7/Epo-S1, progressive apoptosis is induced. In UT7/Epo-S1 cells,this B19V-induced apoptosis has been shown to be associated with expression of the large non-structural protein (NS1) (Sol et al., 1999; Moffatt et al., 1998). The amino acid sequence of

the A6 and V9 NS1 proteins diverge from that of the prototype-encoded counterpart by 6.2%and 6.1%, respectively. The potency of both NS1 proteins of the B19V genotypes 2&3 ininducing apoptosis has not yet been examined.

In the current study, we have used a replication competence system in COS-7 cells tosystematically characterize the transcription profiles of the two B19V genotypes with thecloned nearly-fully length genome of the A6 (genotype-2) and V9 (genotype-3) variants. Wealso investigated the replication competence of the ITRs from the prototype B19V in the contextof the V9 and A6 genomes. Finally, we evaluated the potency of the two novel NS1 proteinsencoded by B19V genotypes 2 and 3 in inducing apoptosis in B19V-permissive cells.

Results

Transcrip tional profiles of h uman erythrovirus es V9 and A6We and others previously showed that the B19V transcription profile in COS-7 cells transfected with a SV40 replication origin (SV40-ori)-containing B19V plasmid closely resembles to thatfrom B19V-infected human bone marrow or erythroid progenitor cells (St et al., 1991; Guanet al., 2008). Therefore, in this study, we used SV40-ori-containing A6 and V9 plasmids(pC1V9 and pC1A6) to transfect COS-7 cells. Replications of these two plasmids in COS-7

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cells were confirmed by Southern blot (data not shown). Transcription profiles were generated from total RNAs isolated from these cells, using RNase protection and Northern blot analysis.

RNase protection analysis of V9 (A6) RNA Total RNA isolated from pC1V9- or pC1A6-transfected COS-7 cells was subjected to RNase protection using probes targeted tothe promoters, intron donor and acceptor sites, and polyadenylation sites. The B19V geneticmap was used as a guide.

V9 (A6) P1 probe Protection with the V9 (A6) P1 probe, which spans the putative P6 promoter, yielded bands of approximately 192 (193) nts and 58 nts, respectively. The 192(193)-nt and 58-nt bands represent RNAs that were transcribed from the P6 promoter and wereunspliced and spliced, respectively, at the first donor site (D1). Thus, the P6 RNA initiationsite of V6 (A6) was located at approximately nt 242 (148), and the D1 donor site was confirmed to be at nt 298 (205). RNA spliced from the D1 donor site accumulated to levels approximately10-times greater than that of RNA unspliced at the D1 site, in the cases of both the V9 and A6RNAs (Fig. 1B&C, lane 1).

V9 (A6) P2 probe Protection with the P2 probe, which covers the putative A1-1 and A1-2acceptors and D2 donor sites, was predicted to yield five bands of 563 (561), 471 (472), 351(352), 274 and 154 nts. The bands at 563 (561) nts, 471 (472) nts and 351 (352) nts, which

protected both the V9 and A6 RNAs from transfected COS-7 cells (Fig. 1B&C, lane 2),represent RNAs that were unspliced and spliced at A1-1 and A1-2, respectively; these RNAswere all unspliced at the D2 donor site. The band at 274 nts represents RNA spliced from theA1-1 acceptor to the D2 donor, and was protected in both V9 and A6 RNA. These resultsconfirmed the A1-1 acceptor and the D2 donor at nt 1802 (1709) and nt 2075 (1982),respectively.

Surprisingly, the 154-nt band that represents RNA spliced at A1-2 acceptor and at the D2 donor was protected in the V9 RNA, but not the A6 RNA (Fig. 1B&C, lane 2), indicating that theA1-2 acceptor may not be utilized significantly during the processing of A6 RNA isolated fromtransfected COS-7 cells. This result thus conformed the A1-2 acceptor of the V9 at nt 1922.To examine further the use of the A1-2 acceptor in processing of the A6 RNA in a B19V-

permissive cell system, we transfected pC1A6 into UT7/Epo-S1 cells. Total RNA was protected with the A6 probe P2; however, a band at 154 nts, which represents RNA spliced atthe A1-2 acceptor and D2 donor, and a band at 352 nts that represents RNA that is spliced atthe A1-2 acceptor but not at the D2 donor were not protected (Fig. 1D, lane 2). In contrast, thecorresponding bands were both protected in V9 RNA isolated from pC1V9-transfected UT7/Epo-S1 cells (Fig. 1D, lane 1). The 352-nt band protected in A6 RNA from COS-7 cells islikely non-specific degraded RNA (Fig. 1C, lane 2), as was confirmed repeatedly by RNase

protection assays (data not shown). These results confirmed that the A1-2 acceptor was notsignificantly utilized in processing A6 RNAs produced in pC1V9-transfected COS-7 and UT7/Epo-S1 cells.

In cells transfected with pC1V9, the levels of RNA spliced at the A1-1 acceptor wereapproximately two-fold greater than those of the RNA spliced at the A1-2 acceptor, which isconsistent with the findings for infection of erythroid progenitors with genotype-1 B19V (Guanet al., 2008).

V9 (A6) P3 probe The P3 probe, which spans the two putative internal polyadenylationsites and the A2-1 acceptor, was protected to yield bands at approximately 665 (668) nts, 424(423) nts, 159 (162) and 133 (124) nts (Fig. 1B&C, lane 3). The 665(668)-nt and 159(162)-nt

bands represent RNAs unspliced and spliced at the A2-1 acceptor, respectively, whichconfirmed the A2-1 acceptor at nt 2937 (2844). The ratio of RNA that was spliced vs unspliced

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at the A2-1 site was approximately 1:6. The 133(124)-nt and 424(423)-nt bands representRNAs that were polyadenylated at the internal polyadenylation sites (pA)p1 and (pA)p2 sites,respectively, which identified the (pA)p1 and (pA)2 sites at nt 2562 (2461) and nt 2853 (2760),respectively. Apparently, about three times more RNA was polyadenylated at the (pA)p1 site.

V9 (A6) P4 and P5 probes The P4 probe, which spans the A2-2 acceptor of the second intron, was protected to yield bands of approximately 243 (244) nts and 80 nts (Fig. 1B&C,

lane 4), and these represent RNAs that were unspliced and spliced RNAs at the A2-2 acceptor site, respectively. This result confirmed the A2-2 acceptor site at nt 4596 (4503). The ratio of RNA that was spliced vs unspliced at the A2-2 was approximately 1:4. The P5 probe, whichspans the distal polyadenylation site, was protected to yield mainly a single band of approximately 190 nts (Fig. 1B&C, lane 5). This indicates that the (pA)d cleavage site is at nt4901 and nt 4808 for the V9 and A6, respectively.

Northern blot analysis of V9 and A6 RNA Northern blot analysis of V9 RNA fromCOS-7 cells exhibited a pattern similar to that of the genotype-1 B19V (Beard et al., 1989; Liuet al., 2004). Hybridization of V9 RNA with the whole NSCap probe revealed four abundantRNA species that accumulated as bands of approximately 4.7 kb, 3.0/3.1 kb, 2.2/2.3 kb and 0.5-0.9 kb (Fig. 2, lane 2). These four bands were also detected following hybridization withthe Cap probe (Fig. 2, lane 6), as well as with the NS probe (Fig. 2, lane 4), suggesting that all

these RNA species were generated from a single promoter located upstream of the V9 genome.An RNA band of approximately 1.8 kb (indicated with an asterisk in Fig. 2, lanes 2, 4 and 6),was always hybridized with the three probes, and the nature of this band is unknown. Takentogether, our findings indicate that the overall profile of V9 transcription is the same as that of genotype-1 B19V.

Hybridization of A6 RNA with the whole NSCap probe revealed four abundant RNA speciessimilar to those from V9 RNA, i.e. 4.7 kb, 3.1 kb, 2.3 kb and 0.5-0.9 kb bands (Fig. 2, lane 3).These four bands were also detected following hybridization with the Cap probe (Fig. 2, lane7). However, when it came to hybridization with the NS probe, unlike the V9 RNA, the A6RNA did not produce small RNA bands at 0.5-0.9 kb. It is likely that the V9 NS probehybridized poorly with these small RNAs, which were spliced at the D1 donor and thuscontained a small exon ( 57 nts), due to sequence variation between V9 and A6.

Combining the results obtained from RNase protection assay and Northern blot analysisenabled us to establish the transcription maps for V9 and A6, respectively, as shown in Fig. 3.In general, alternative processing of the V9 and A6 RNAs from transfected COS-7 cellsgenerated different RNA species that are required to produce the non-structural proteins, i.e.

NS1, 8-kDa and 10-kDa, and the structural proteins, VP1 and VP2. In the case of A6 RNA processing, the failure of splicing at the A1-2 acceptor does not prevent generation of sufficientmRNA species encoding for these viral proteins. The R1 , R2 and R3 mRNAs of the V9 RNAand the R1 and R2 mRNAs of the A6 RNA were polyadenylated at the (pA)p2 site and did not appear clearly on Northern blots, indicating that they are likely minor transcripts. Ingenotype-1 B19V, RNA polyadenylated at the (pA)p2 accounts for only 10% of the RNA thatis internally polyadenylated (Yoto et al., 2006). The 4.7 kb R0 mRNA present on maps of V9and A6 is the full-length transcript that reads through the (pA)p sites. It was detected ingenotype-1 RNA from transfected cells (Liu et al., 2004), but not in genotype-1 RNA fromvirus-infected cells (Ozawa et al., 1987). Since the genotype-1 RNA profile generated in COS-7cells following transfection with a replication-competent construct is similar to that followinginfection with genotype-1 virus (St et al., 1991;Yoto et al., 2006;Guan et al., 2008), we believethat the transcription profiles of A6 and V9 during virus infection are similar to those observed in transfected cells.

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In B19V-permiss ive UT7/Epo-S1 cells, B19V genoty pe-1 ITRs support repli cation of th eincomplete V9 genome, which p roduces infectiou s progeny vi rus, but not o f the A6 genome

The terminal repeats of both genotype-2 and genotype-3 have not been characterized; therefore,we next examined whether the prototype-1 terminal repeats can support replication of the A6or the V9 incomplete genome in UT7/Epo-S1 cells. To this end, we constructed chimericgenomes of B19VITR-V9 and B19VITR-A6, as diagramed in Fig. 4A, by inserting the P6-driven NS1- and Cap-encoding region ( NSCap ) of the two variants into vectors bearing theB19V ITRs derived from an infectious clone of genotype-1, pB19-M20 (Zhi et al., 2004).Excised B19V DNAs were transfected into UT7/Epo-S1 cells and low molecular-weight DNAwas extracted for Southern blot analysis. B19V DNA replication can be identified by the

presence of a DpnI-digestion-resistant DNA band. As shown in Fig. 4B, the prototypic ITRssupported replication of the V9 NSCap, but not the A6 NSCap (Fig. 4B, lanes 8&10,respectively), suggesting that the A6 NS1 protein may contain mutations that preventreplication of the genotype-1 ITRs-containing A6 genome. The positive control (the infectiousgenotype-1 DNA, M20) generated significant quantities of DpnI-resistant DNA (Fig. 4B, lane4). In contrast, the negative control [the NS1-knock-out mutant, M20NS1(-)], did not producea DpnI-resistant band (Fig. 4B, lane 6). NS1 expression was confirmed in cells transfected withthe M20, B19VITR-V9 and B19VITR-A6 DNAs, but not in M20NS1(-)-transfected cells (datanot shown). Based on these findings, we predict that the ITR structure of the A6 variant mustdiffer from that of the prototypic ITR.

We next set out to determine whether transfecting UT7/Epo-S1 cells with B19VITR-V9 or B19VITR-A6 leads the production of infectious progeny virus. To this end, we transfected UT7/Epo-S1 with B19VITR-V9 or B19VITR-A6 and used lysates of these cells to infectB19V-permissive ex vivo-expanded CD36 + erythroid progenitor cells (CD36 + EPCs) (Wonget al., 2008; Guan et al., 2008). Lysates generated from UT7/Epo-S1 cells transfected withM20 DNA and M20NS(-) served as a positive and a negative control, respectively. The

production of progeny virus was assayed by the infectivity of CD36 + EPCs, and quantified byreverse-transcription (RT)-real time PCR analysis of spliced VP2 and 11-/10-kDa viral mRNAs

produced in infected CD36 + EPCs. The infectivity of the progeny virus obtained from lysatesof B19VITR-V9-transfected UT7/Epo-S1 cells was nearly equivalent to that of virus in lysatesof M20-transfected (positive control) cells. In contrast, infectivity of the progeny virus fromlysates from B19VITR-A6-transfected UT7/Epo-S1 cells and M20NS(-) (negative control) -transfected cells, could not be detected (Fig. 4C). These results further confirmed that, in UT7/Epo-S1 cells, replication competence and progeny virus production of the V9 genome issupported by the genotype-1 B19V ITRs, but that is not the case for the A6 genome.

The V9 and A6 NS1 protein s are potent inducers of apoptosi s in UT7/Epo-S1 cells

B19V infection induces cell death through an apoptotic pathway, in both primary erythroid cells and B19V-permissive cell lines (Sol et al., 1999; Moffatt et al., 1998). This cell death has

been attributed to the NS1 protein. Apoptosis has been reported to be induced in the NS1-expressing UT7/Epo-S1 as well as in NS1-transfected cells (Sol et al., 1999; Moffatt et al.,1998; Poole et al., 2004). Given that the A6 and V9 NS1 proteins diverge approximately 6.0from that of the genotype-1, we decided to examine the pro-apoptotic nature of these two novel

NS1 proteins. After initiating apoptosis, cells translocate the membrane phosphatidylserinefrom the inner face of the plasma membrane to the cell surface, and this event can be easilydetected by staining with a FITC-conjugated AnnexinV protein. AnnexinV staining requiresthe use of living cells. To sort NS1-expressing cells, we transfected GFP-fused V9 and A6

NS1constructs into UT7/Epo-S1 cells. Two days after transfection, cells then were stained alive with AnnexinV and PI and were analyzed by flow cytometry. GFP-fused genotype-1 NS1and GFP alone were used as positive and negative controls, respectively. Our results showed that both NS1 proteins of the A6 and V9 variants behaved much like the genotype-1 B19V

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NS1, inducing a significant percentage of the GFP-positive (GFP+) cells in the population of transfected cells to also become AnnexinV-positive compared with the GFP control (Fig. 5).However, in GFP negative [GFP(-)] cells, the population of AnnexinV+ among thosetransfected with the genotype-1, A6, and V9 GFP-NS1 constructs did not differ significantlyfrom that of the GFP transfection control. More specifically, in the GFP+ cell population,transfection of the genotype-1, V9 and A6 GFP-NS1 constructs induced AnnexinV+/PI-

populations of 21.1%, 22.6% and 22.9%, respectively (cells at early stages of apoptosis) (Fig.

5). In the same population, genotype-1, V9 and A6 GFP-NS1 induced an AnnexinV+/PI+ population of 17.2%, 14.3% and 12.9%, respectively (cells at late stages of apoptosis) (Fig. 5).The GFP control only induced an AnnexinV+/PI- and an AnnexinV+/PI+ populations of 12.8%and 4.6%, respectively (Fig. 5). We thus conclude that both NS1 proteins of the genotypes 2and 3 have a potency similar to that of the prototype NS1 to induce apoptosis in UT7/Epo-S1cells.

Discussion

Recently, human parvovirus B19V has been described to diverge genetically (Hokynar et al.,2007). However, serological studies of the three genotypes of B19V confirmed that they belongto one serotype (Ekman et al., 2007); and all the three genotypes show a tropism for erythroid cells (Hokynar et al., 2007). Since the transcription profile of prototype B19V generated by

transfecting COS-7 cells is similar to that during virus infection of B19V-permissive cells(Ozawa et al., 1987; Beard et al., 1989; Liu et al., 2004; Yoto et al., 2006), we believe that thetranscriptional profiles of V9 and A6 variants presented in this study resemble those duringvirus infection. A unique feature we have identified is that the genotype-2 A6 variant used onlyone splice acceptor to remove the first intron. However, the overall RNA profile of thegenotype-3 V9 variant is the similar as that of the prototype B19V.

Prototype B19V replicates exclusively in erythroid progenitors of human bone marrow, and produces extraordinarily high virus yields in blood during the early phase of infection(Anderson et al., 1985; Enders et al., 2006; Ozawa et al., 1986). In contrast, high-titers of genotype-2 and genotype-3 are rarely identified (Blumel et al., 2005; Liefeldt et al., 2005;

Nguyen et al., 1999; Nguyen et al., 2002; Servant et al., 2002). More importantly, thegenotype-2 variant often persistently presents in skin; and genotype-3 has been suggested to

have remained absent from wide circulation in Europe (Norja et al., 2006). In the current study,we have shown that the genotype-2 A6 variant uses only the first acceptor to remove the firstintron, and therefore fails to produce the corresponding R5, R7 and R9 mRNAs (Fig. 3B).Although the R4, R6 and R8 mRNAs encode VP1, VP2 and 10-kDa, respectively, all three aredi-cistronic and in them, the 8-kDa ORF is located upstream of the VP1/VP2 ORF and the 10-kDa ORF, respectively. It is thus possible that these di-cistronic mRNAs reduce the translationefficiency of VP1/VP2 and 10-kDa. In addition, the inactive A1-2 acceptor site could decreasethe overall number of RNAs spliced at the D2 donor and, in turn, reduces the levels of proteinexpression during active virus replication. Thus, we hypothesize that the inactive A1-2 acceptor is probably a disadvantage for the processing of A6 virus mRNAs as well as a disadvantagefor protein translation, which could possibly maintain virus production at a low level.

Sequence alignments among members of all three genotypes (nt 2139-2241 with respect to the prototype B19V J35 isolate, Genbank accession no.: AY386330) showed that only four variations present in the A1-2 acceptor region. Between V9 and A6, these nucleotides haveonly one difference, an A versus a G at nt 2179. Interestingly, the position of this nucleotidesuggests that it could be the branch point for the A1-2 acceptor. However, mutation of the Gto A in A6 genome did not rescue splicing at the A1-2 site (data not shown), suggesting thatefficient splicing at the A1-2 site may require an exon and/or intron splicing enhancer. In fact,the three genotypes vary by approximately 12% in the region spanning the D2 donor (nt

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2305-2581), which supports that variations in the potential splicing enhancers may affectsplicing at the A1-2 acceptor site. We realized that the conclusion that the genotype-2 A6 used only one splice acceptor to remove the first intron was drawn only from results obtained bytransfection of the cloned genome of A6 variant. However, an A6 mutant that bears an Amutation at nt 2180, which reverts to an identical sequence from nt 1800 to nt 2500 to that of the Lali variant (Hokynar et al., 2002), did not rescue splicing at the A1-2 (data not shown).Further investigation into whether the A1-2 acceptor is used with genomes of other genotype-2

and their viruses, such as the IM-81 (Blumel et al., 2005), is required to demonstrate the biological importance of the inactive A1-2 acceptor.

The NS1 proteins of the three genotypes diverge significantly. The genotype-2 LaLi and genotype-3 V9 variants diverge nearly 13% from in the prototype B19V isolate within the NS1-encoding sequence (Hokynar et al., 2002). On the protein level, however, the LaLi, A6 and V9variants are only 6.0% divergent from the B19V prototype. The B19V NS1 protein is amultifunctional protein. Parvovirus NS1 contains a DNA binding domain at its N-terminus,the conserved motifs for single-strand nicking activity and an ATP/helicase domain at thecenter, and a transcription activation domain at the C-terminus (Cotmore and Tattersall,2006). The genotype-1 NS1 has been found to enhance equally on promoters of all threegenotypes (Ekman et al., 2007). We have shown in this study that the genotype-2 A6 NS1 doesnot support replication of the genotype-1 ITR-based A6- NSCap genome (Fig. 4.), suggesting

that the 6% divergence either is sufficient to alter the specific binding of the A6 NS1 to thegenotype-1 ITR, or to reduce the nicking activity at the terminal resolution site. Interestingly,in a similar genotype-1 ITR-based V9- NSCap genome, the V9 NS1 supported not onlygenotype-1 ITR-dependent replication, but also progeny virus production in UT7/Epo-S1 cells.Thus, the V9 NS1 is fully functional with respect to enabling replication of the B19VITR-V9genome, as well as synthesis of the single-stranded DNA genome and its packaging. Therefore,we have shown, for the first time, that the divergent B19V NS1 proteins have genotype-specificactivity in DNA replication. We hypothesize that the ITR structure of the A6 variant must differ from that of the genotype-1. Whether the NS1 of other genotype-2 variants is similar to the A6

NS1, and whether genotype-2 variants share a common ITR structure, require further investigation.

Our results also have confirmed that both the A6 and V9 NS1 proteins possess a similar potency

in inducing apoptosis in B19V-permissive cells, indicating that the NS1 proteins of B19Vgenotypes 2 and 3 have the same function in the pathogenesis of virus infection as the prototype

NS1. The prototypic NS1-induced cytoxicity and apoptosis of infected erythroid progenitorscause the disease outcomes characteristic of B19V infection (Brown and Young, 1997; Moffattet al., 1998; Sol et al., 1999).

Materials and MethodsCells

COS-7 cells (CRL-1651; ATCC) were maintained in Dulbecco's modified Eagle's medium(DMEM) with 10% fetal calf serum (FCS) at 37C in 5% CO 2. UT7/Epo-S1 cells were cultured in DMEM with 10% FCS and 2 units/ml of Epo [Procrit (Epoetin Alfa), Centocor Ortho BiotechInc., Horsham, PA] at 37C in 5% CO 2. The process of generating of primary human CD36 +

erythroid progenitor cells (CD36 + EPCs) has been described elsewhere (Guan et al., 2008;Wong et al., 2008).

Transfection

Per 60-mm dish, 2 g of DNA were transfected into COS-7 cells, using Lipofectamine and Plus reagents (Invitrogen) as previously described (Qiu and Pintel, 2002). UT7/Epo-S1 cells

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were transfected with 2 g of DNA per 2 10 6 cells using a universal reagent (DNAproject,WA) and program X005 using the Nucleofector (Lonza, MD).

Plasmid construction

(i) pC1V9 and pC1A6 plasmids: The original A6 plasmid, a pCR-Blunt II TOPO (Invitrogen)vector into which the full-length coding sequence of A6 (4844 bp) without the 3 - and 5 -ITRs(Genbank accession no.: AY064475) had been cloned, was a gift from Dr. Kevin Brown.Plasmid pC1A6 was constructed by replacing the B19V NSCap gene with the A6 sequence of nt 1-4844 in pC1NS1(-) (Yoto et al., 2006). The pC1 backbone was constructed by removingthe CMV-GFP expression cassette (nt 8-1477) from pEGFP-C1 (Clontech). A SV40-ori isincluded in pC1.

The V9-C22 plasmid, consisting of the entire V9 genome except the 3 - and 5 -ITRs (5028 bp,Genbank accession no.: AJ249437), was obtained from Collection National deCulture deMicroorganisms (Institute Pasteur, Paris, France: CNCM1-2066) as a transferred material.

pC1V9 was constructed by replacing the B19V NSCap gene with the V9 sequenceencompassing nt 1-5028 in pC1NS1(-).

(ii) pB19VITR-V9 and pB19VITR-A6 plasmids: All the nucleotide numbers of thegeneotype-1 B19V refer to the J35 isolate (Genbank accession no.: AY386330). To insert theA6 NSCap gene into the J35-ITRs from pM20 (an infectious clone of B19), we first modified the pN8 (Zhi et al., 2004) to contain the B19V sequence of nt 182-5412, including two half-ITRs with a BssHII site, which was named pBssH-N8. Then, the B19V sequences of nt383-5209 and nt 383-5210 on pBssH-N8 were replaced with the corresponding V9 sequence(nt 105-4919) and A6 sequence (nt 12-4827), respectively, which resulted in the plasmids

pBssH-V9 and pBssH-A6. The BssHII-digested large fragments of pBssH-V9 and pBssH-A6were inserted into BssHII-digested pM20, which resulted in the final plasmids pB19VITR-V9and pB19VITR-A6, respectively. A schematic diagram of the excised DNAs of B19VITR-A6and B19VITR-V9 is shown in Fig. 4A.

(iii) GFP-fused NS1 constructs: The NS1 ORFs of V9 (nt 328-2340), A6 (nt 235-2247) and B19V (nt 616-2628) were inserted into BamHI-XhoI digested pcDNAGFP vector,respectively, to construct the pGFP-V9NS1, pGFP-A6NS1 and pGFP-B19VNS1, respectively.The pcDNA-GFP was made by inserting the GFP-coding sequence into the HindIII/BamHIdigested pcDNA3 (Invitrogen).

(iV) Clones used to generate probes for RNase protection: To map the transcription unitsof V9 and A6, we used probes V9 (A6) P1, P2, P3, P4 and P5. These probes were constructed

by cloning the following regions of V9 (A6) into BamHI-HindIII digested pGEM3Z(Promega): nt 193-432 (100-340) [V9 (A6) P1], nt 1710-2272 (1620-2180) [V9 (A6) P2], nt2430-3095 (2338-3005) [V9 (A6) P3], nt 4433-4675 (4339-4582) [V9 (A6) P4] and nt4712-4941 (4619-4844) [V9 (A6) P5].

RNA iso lation, RNase protection and Northern b lot analysis

Total RNA was isolated from transfected cells two days post transfection using TRIZOLreagent (Invitrogen).

An RNase protection assay was performed essentially as previously described (Naeger et al.,1992; Schoborg and Pintel, 1991). Probes were generated from BamHI-digested templates byin vitro transcription with Sp6 polymerase using the MAXIscript kit (Ambion) and followingthe manufacture's instruction. RNA hybridizations for RNase protection were done in the

presence of a substantial excess of probe, and signals were quantified with the Storm 856

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phosphor imager and Image Quant TL software v2005 (GE Healthcare). Relative molar ratiosof individual RNA species were determined after adjusting for the number of 32P-labeled uridines (U) in each protected fragment as previously described (Schoborg and Pintel, 1991).

Northern blot analyses were performed as previously described (Pintel et al., 1983; Qiu et al.,2002), using total RNA samples and 32P-labeled DNA probes as indicated. All Northern probeswere digested from corresponding plasmids and are diagramed in Fig. 3.

Southern blot analysis

At two days after transfection, cells were collected and washed twice with phosphate-buffered saline. Isolation of low molecular-weight DNA (Hirt DNA) and Southern blotting were

performed essentially as described previously (Guan et al., 2008). Blots were hybridized withthe V9 NSCap probe (nt 1-5028) (Fig. 3), and signals were developed by exposing the blots toX-ray film.

Virus generation and i nfection

Transfected UT7/Epo-S1 cells were harvested three days after transfection. After the cells werefrozen and thawed three times, supernatant was collected. 200 l of the supernatant wereincubated with 2 10 5 of CD36 + EPCs with a slow rotation, at 4C for 1.5 hrs. Infected cells

were pelted by low-speed centrifugation, and were cultured at a concentration of 2 105

cells/ml at 37 C with 5% CO 2. At three days postinfection, mRNA was directly isolated from thecells, using the TurboCapture 8 mRNA kit (Qiagen) according to the manufacturer'sinstructions.

Reverse transcr iption (RT) and real time-PCR

Isolated mRNA was reverse-transcribed to cDNA using random hexamers (Promega) and MMLV-RT (Invitrogen), following the manufacturer's instructions. cDNA copy number wasdetermined by real-time PCR as described previously (Guan et al., 2008; Guan et al., 2009),using the TaqMan universal PCR master mix (Takara).

The forward and reverse primers and probe used in real-time PCR for cDNAs converted fromthe respective genotype-1 VP2 and 11kDa mRNAs were described previously (Guan et al.,

2009). The probes used for cDNAs converted from A6 VP2 and 10-kDa mRNAs, were thesame as those used for prototype B19V, respectively, and the forward and reverse primers used were as follows: for VP2 mRNA: 5 -GACCAGTTCAGGAGAATCAT-3 (nt 1875-1894), 5 -TTCTGAGGC GTTGTATGC-3 (nt 2911-2894); for 10-kDa mRNA: 5 -GAAGCCTTTTACACTCCACTTG-3 (nt 1942-1963), 5 -TGGCAGTCCACAATTCTTCAG G-3 (nt 4565-4544). The probe used for cDNAs converted from V9 VP2 mRNA was the same for prototype VP2 mRNA, and the probe used for cDNAconverted from V9 10-kDa mRNA were as the following: 5 FAM-CGATCAGTTTCGTGAACT/CTACAGATGGA-3 BHQ (D2/A2-2). The forward and reverse primers used for cDNAs converted from V9 VP2 and 11kDa mRNAs were as follows: for VP2 mRNA:5-5-GACCAGTTCAGGAGAATCAT-3 (nt 1968-1988)-3 , 5-TTCTGAGGCGTTGTACGC-3 (nt 3004-2987); for 10-kDa mRNA: 5 -GAAGCTTTTTACACGCTT G-3 (nt 2035-2056), 5 -TGGCAG TCCACAATTCTTCAGG-3 (nt 4568-4637).

All the splice donor (D2) and acceptor (A2-1 and A2-2) sites for prototype B19V weredescribed as previously (Guan et al., 2008), and those for A6 and V9 are described in Fig. 3.

cDNA copies of -actin mRNA were quantified using the forward and reverse primers and probe as previously reported using JOE-labeled TaqMan probe (Kammula et al., 1999).

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Standard curves for -actin, VP2 and 11-/10-kDa cDNAs were generated from serial dilutionsof a -actin cDNA plasmid (Wong et al., 2008) and the B19V, V9 and A6 VP2 cDNA and 11-/10-kDa cDNA plasmids. The copy numbers of all B19V mRNAs are presented as copies per l of the cDNA reaction mixture, and were normalized with respect to the copy number of the-actin mRNA produced in the same reaction.

Flow cytometry analysis Cells were double-stained live with Cy5-conjugated AnnexinV

(BD Biosciences) and Propidium Iodide (PI, Sigma) in order to detect apoptotic cells. Stainingwas carried out according to the manufacturer's instructions (BD Biosciences). All sampleswere analyzed on the three-laser flow cytometer (LSR II, BD Biosciences) at the FlowCytometry Core of the University Kansas Medical Center. Flow cytometry data were analyzed using FACS DIVA software (BD Biosciences).

AcknowledgmentsThis work was supported by PHS grant RO1 AI070723 from NIAIDand grant P20 RR016443 from the NCRR COBRE

program. We thank Dr. Kevin Brown for providing valuable reagents.

ReferencesAllander T, Tammi MT, Eriksson M, Bjerkner A, Tiveljung-Lindell A, Andersson B. Cloning of a human

parvovirus by molecular screening of respiratory tract samples. Proc Natl Acad Sci U S A 2005;102(36):1289112896. [PubMed: 16118271]

Anderson, MJ.; Jones, SE.; Minson, AC. DNA J Med Virol. Vol. 15. 1985. Diagnosis of human parvovirusinfection by dot-blot hybridization using cloned viral; p. 163-172.

Beard C, St AJ, Astell CR. Transient expression of B19 parvovirus gene products in COS-7 cellstransfected with B19-SV40 hybrid vectors. Virology 1989;172(2):659664. [PubMed: 2800343]

Blumel J, Eis-Hubinger AM, Stuhler A, Bonsch C, Gessner M, Lower J. Characterization of ParvovirusB19 genotype 2 in KU812Ep6 cells. J Virol 2005;79(22):1419714206. [PubMed: 16254355]

Brown, KE.; Young, N. Human parvovirus B19: Pathogenesis of disease. In: Anderson, LJ.; Young, N.,editors. Human parvovirus B19. Basel, Switzerland: Karger; 1997. p. 105-119.

Candotti D, Etiz N, Parsyan A, Allain JP. Identification and characterization of persistent humanerythrovirus infection in blood donor samples. J Virol 2004;78(22):1216912178. [PubMed:15507603]

Cotmore, SF.; Tattersall, P. A rolling-haipin strategy: basic mechanisms of DNA replication in the parvoviruses. In: Kerr, J.; Cotmore, SF.; Bloom, ME.; Linden, RM.; Parrish, CR., editors.Parvoviruses. London: Hoddler Arond; 2006. p. 171-181.

Ekman A, Hokynar K, Kakkola L, Kantola K, Hedman L, Bonden H, Gessner M, Aberham C, Norja P,Miettinen S, Hedman K, Soderlund-Venermo M. Biological and immunological relations amonghuman parvovirus B19 genotypes 1 to 3. J Virol 2007;81(13):69276935. [PubMed: 17409158]

Enders M, Schalasta G, Baisch C, Weidner A, Pukkila L, Kaikkonen L, Lankinen H, Hedman L,Soderlund-Venermo M, Hedman K. Human parvovirus B19 infection during pregnancy--value of modern molecular and serological diagnostics. J Clin Virol 2006;35(4):400406. [PubMed: 16332455]

Guan W, Cheng F, Yoto Y, Kleiboeker S, Wong S, Zhi N, Pintel DJ, Qiu J. Block to the production of full-length B19 virus transcripts by internal polyadenylation is overcome by replication of the viralgenome. J Virol 2008;82(20):99519963. [PubMed: 18684834]

Guan W, Wong S, Zhi N, Qiu J. The genome of human parvovirus B19 virus can replicate in non- permissive cells with the help of adenovirus genes and produces infectious virus. J Virol 2009;83(18):95419553. [PubMed: 19587029]

Hokynar K, Norja P, Hedman K, Soderlund-Venermo M. Tissue persistence and prevalence of B19 virustypes 13. Future Virology 2007;2(4):377388.

Hokynar K, Soderlund-Venermo M, Pesonen M, Ranki A, Kiviluoto O, Partio EK, Hedman K. A new parvovirus genotype persistent in human skin. Virology 2002;302(2):224228. [PubMed: 12441066]

Chen et al. Page 10


NI H-P A A



NI H-P A A ut h or

Manus c r i pt

8/14/2019 Ni Hms 160986

11/18

Kammula US, Lee KH, Riker AI, Wang E, Ohnmacht GA, Rosenberg SA, Marincola FM. Functionalanalysis of antigen-specific T lymphocytes by serial measurement of gene expression in peripheral

blood mononuclear cells and tumor specimens. J Immunol 1999;163(12):68676875. [PubMed:10586088]

Liefeldt L, Plentz A, Klempa B, Kershaw O, Endres AS, Raab U, Neumayer HH, Meisel H, Modrow S.Recurrent high level parvovirus B19/genotype 2 viremia in a renal transplant recipient analyzed byreal-time PCR for simultaneous detection of genotypes 1 to 3. J Med Virol 2005;75(1):161169.[PubMed: 15543575]

Liu Z, Qiu J, Cheng F, Chu Y, Yoto Y, O'Sullivan MG, Brown KE, Pintel DJ. Comparison of thetranscription profile of simian parvovirus with that of the human erythrovirus B19 reveals a number of unique features. J Virol 2004;78(23):1292912939. [PubMed: 15542645]

Moffatt S, Yaegashi N, Tada K, Tanaka N, Sugamura K. Human parvovirus B19 nonstructural (NS1) protein induces apoptosis in erythroid lineage cells. J Virol 1998;72(4):30183028. [PubMed:9525624]

Naeger LK, Schoborg RV, Zhao Q, Tullis GE, Pintel DJ. Nonsense mutations inhibit splicing of MVMRNA in cis when they interrupt the reading frame of either exon of the final spliced product. GenesDev 1992;6(6):11071119. [PubMed: 1592259]

Nguyen QT, Sifer C, Schneider V, Allaume X, Servant A, Bernaudin F, Auguste V, Garbarg-Chenon A. Novel human erythrovirus associated with transient aplastic anemia. J Clin Microbiol 1999;37(8):24832487. [PubMed: 10405389]

Nguyen QT, Wong S, Heegaard ED, Brown KE. Identification and characterization of a second novelhuman erythrovirus variant, A6. Virology 2002;301(2):374380. [PubMed: 12359439]

Norja, P.; Hokynar, K.; Aaltonen, LM.; Chen, R.; Ranki, A.; Partio, EK.; Kiviluoto, O.; Davidkin, I.;Leivo, T.; Eis-Hubinger, AM.; Schneider, B.; Fischer, HP.; Tolba, R.; Vapalahti, O.; Vaheri, A.;Soderlund-Venermo, M.; Hedman, K. Proc Natl Acad Sci U S A. Vol. 103. 2006. Bioportfolio:lifelong persistence of variant and prototypic erythrovirus DNA genomes in human tissue; p.7450-7453.

Ozawa K, Ayub J, Hao YS, Kurtzman G, Shimada T, Young N. Novel transcription map for the B19(human) pathogenic parvovirus. J Virol 1987;61(8):23952406. [PubMed: 3599180]

Ozawa K, Kurtzman G, Young N. Replication of the B19 parvovirus in human bone marrow cell cultures.Science 1986;233(4766):883886. [PubMed: 3738514]

Pintel D, Dadachanji D, Astell CR, Ward DC. The genome of minute virus of mice, an autonomous parvovirus, encodes two overlapping transcription units. Nucleic Acids Res 1983;11(4):10191038.[PubMed: 6828378]

Poole BD, Karetnyi YV, Naides SJ. Parvovirus B19-induced apoptosis of hepatocytes. J Virol 2004;78(14):77757783. [PubMed: 15220451]

Qiu J, Nayak R, Tullis GE, Pintel DJ. Characterization of the transcription profile of adeno-associated virus type 5 reveals a number of unique features compared to previously characterized adeno-associated viruses. J Virol 2002;76(24):1243512447. [PubMed: 12438569]

Qiu J, Pintel DJ. The adeno-associated virus type 2 Rep protein regulates RNA processing via interactionwith the transcription template. Mol Cell Biol 2002;22(11):36393652. [PubMed: 11997501]

Sanabani S, Neto WK, Pereira J, Sabino EC. Sequence variability of human erythroviruses present in bone marrow of Brazilian patients with various parvovirus B19-related hematological symptoms. JClin Microbiol 2006;44(2):604606. [PubMed: 16455922]

Schoborg RV, Pintel DJ. Accumulation of MVM gene products is differentially regulated by transcriptioninitiation, RNA processing and protein stability. Virology 1991;181(1):2234. [PubMed: 1825251]

Servant A, Laperche S, Lallemand F, Marinho V, De Saint MG, Meritet JF, Garbarg-Chenon A. Geneticdiversity within human erythroviruses: identification of three genotypes. J Virol 2002;76(18):9124 9134. [PubMed: 12186896]

Sol N, Le JJ, Vassias I, Freyssinier JM, Thomas A, Prigent AF, Rudkin BB, Fichelson S, Morinet F.Possible interactions between the NS-1 protein and tumor necrosis factor alpha pathways in erythroid cell apoptosis induced by human parvovirus B19. J Virol 1999;73(10):87628770. [PubMed:10482630]

Chen et al. Page 11


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Srivastava A, Lu L. Replication of B19 parvovirus in highly enriched hematopoietic progenitor cells fromnormal human bone marrow. J Virol 1988;62(8):30593063. [PubMed: 3392774]

St AJ, Beard C, Humphries K, Astell CR. Analysis of splice junctions and in vitro and in vivo translation potential of the small, abundant B19 parvovirus RNAs. Virology 1991;183(1):133142. [PubMed:2053277]

Wong S, Zhi N, Filippone C, Keyvanfar K, Kajigaya S, Brown KE, Young NS. Ex vivo-generated CD36+ erythroid progenitors are highly permissive to human parvovirus B19 replication. J Virol 2008;82(5):24702476. [PubMed: 18160440]

Yoto Y, Qiu J, Pintel DJ. Identification and characterization of two internal cleavage and polyadenylationsites of parvovirus B19. RNA J Virol 2006;80(3):16041609.

Young NS, Brown KE. Parvovirus B19. N Engl J Med 2004;350(6):586597. [PubMed: 14762186]Zhi N, Zadori Z, Brown KE, Tijssen P. Construction and sequencing of an infectious clone of the human

parvovirus B19. Virology 2004;318(1):142152. [PubMed: 14972543]

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Figure 1. Transcription mapping of V9 and A6 RNA by RNase protection assay (RPA)(A) Schematic diagram of the V9 (A6) genome and the probes used for RPA The landmarksof transcription: the promoter (P6), the splice donor sites (D1 and D2), the acceptor sites (A1-1,A1-2, A2-1 and A2-2), the internal polyadenylation signal (pA)p and the distal polyadenylationsits (pA)d, are indicated. The RPA probes V9 (A6) P1, V9 (A6) P2, V9 (A6) P3, V9 (A6) P4and V9 (A6) P5 are shown with their respective V9 nucleotide numbers with the A6 nucleotidenumbers in parentheses, along with the designated bands they are expected to protect and their

predicted sizes. Spl, spliced RNAs; Unspl, unspliced RNAs. (B&C) Mapping of the V9 (B)and A6 (C) transcription units by RPA. 10 g of total RNA isolated two days after thetransfection of COS-7 cells with plasmid pC1V9 (B) or pC1A6 (C) were protected by V9 (B)or A6 (C) probes P1, P2, P3, P4 and P5. Lane 1, 32P-labeled RNA markers with sizes indicated.The sizes of the protected bands in the lanes are indicated. (D) RPA of V9 (A6) RNA isolatedfrom transfected UT7/Epo-S1 cells. 10 g of total RNA isolated two days after transfectionof UT7/Epo-S1 cells with the plasmid pC1V9 or pC1A6 was protected by the V9 or A6 probeP2. The sizes of the protected bands in the lanes are indicated. Arrow heads indicate the absenceof A6 RNAs spliced at the A1-2 site. Asterisks indicate undigested probe bands.

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Figure 2. Northern blot analysis of V9 and A6 RNATotal RNA isolated from pC1V9- and pC1A6- transfected COS-7 cells two days post-transfection was used for Northern blot analysis. The blot was hybridized to three V9 DNA

probes ( NSCap , NS and Cap ), which span various regions of the V9 genome, as indicated. The probes used are diagramed at the bottom of Fig. 3A. RNA bands detected by each probe areindicated by their respective sizes in kb to the right side of each blot. Asterisks indicate bandsof unknown identity. RNA maker ladder (Ambion) is shown in lane 1.

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Figure 3. Transcription maps of V9 and A6The V9 and A6 genomes in panels A and B, respectively, are shown to scale with transcriptionlandmarks confirmed by RPA [i.e. the P6 promoter, the splice donors (D), and acceptors (A)

sties, the (pA)p and the (pA)d]. All of the RNA species detected are diagrammed, with their sizes in absence of the polyA tail indicated. The ORFs that each encodes [with reference tothose shown in the B19V map (Ozawa et al., 1987; Guan et al., 2008)] are also diagramed, and the predicted sizes (kDa) of the translated proteins are indicated.

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Figure 4. Replication of the V9 and A6 NSCap genomes in the presence of genotype-1 ITRs(A) Schematic diagrams of B19V DNAs B19V DNA M20 is the duplex replicative form (RF)

of the genotype-1 genome, which is able to replicate and produce progeny virus in B19V- permissive cells (Zhi et al., 2004). The bubbles within each ITR reflect potential inter-strand folding. The position of the P6 promoter, as well as those of the ORFs for the five B19V

proteins, are also indicated. The prototype B19V ITRs were fused to both ends of the V9 and A6 genomes, at nucleotide numbers shown as ITR nucleotide number/(V9 or A6 nucleotidenumber), under the diagram of each chimeric construct (B19VITR-V9 or B19VITR-A6).BssHII sites were used to clone the V9 or A6 genome into the prototype B19V ITRs, asdescribed in the Materials and Methods section. (B) Southern blot analysis. Hirt DNA isolated

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from excised B19VITR-V9- or B19VITR-A6-transfected UT7/Epo-S1 cells two days post-transfection was digested with EcoRI and DpnI which are indicated as DpnI (-) and DpnI+samples, respectively, and these samples were used in Southern blot analysis. Hirt DNAsamples isolated from B19V M20- and M20NS1(-)-transfected UT7/Epo-S1cells were used as positive and negative controls, respectively, of B19V DNA replication. The blot washybridized to the B19V NSCap DNA probe (Guan et al., 2008). EcoRI-digested and DpnI-digested M20 DNA (6 ng) were run in lane 1 and lane 2, respectively, and were used as a

control of DpnI digestion and a DNA size maker. (C) Quantification of progeny virusproduction. UT7/Epo-S1 cells were transfected with four B19V DNAs, as shown in panel A.Cell lysates prepared at three days post-transfection were used to infect CD36 + EPCs, and theinfectivity of these cell lysates was quantified by a RT-real-time-PCR strategy that detected specifically the VP2 and 11-/10-kDa mRNAs in the CD36 + EPCs lysates. In each sample, theabsolute number of mRNA copies was normalized to the level of -actin mRNA (10 4 copies

per l). Results shown represent the average and standard deviation for data from at least threeindependent experiments. UD denotes undetectable.

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Figure 5. Transfection of V9 and A6 NS1 induces apoptosis in B19V-permissive cellsUT7/Epo-S1 cells were transfected with pGFP (as negative control), pGFP-B19VNS1 (positivecontrol), pGFP-V9NS1 and pGFP-A6NS1. Cells were double-stained with AnnexinV and

propidium iodide (PI) at two days post-transfection, and were then subjected to flow cytometry.Both GFP-negative [GFP(-)] and GFP-positive (GFP+) cell populations were gated to plot cells

by PI staining vs AnnexinV staining. A representative experiment of three is shown. Theaverage percentages with standard deviation of the AnnexinV+/PI+ and AnnexinV+/PI-

populations are shown in the upper right and lower right quadrants, respectively.

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